This study aimed to develop biocontrol Bacillus and explore bacterial biocontrol substances. According to the blood agar test, strain FJAT-14262 was screened as a biosurfactant-producer. The biosurfactant-producing ability of FJAT-14262 was further confirmed by the oil spreading tests because of its amphipathic character. Furthermore, its fermentation supernatant could decrease the surface tension from 74.1 to 32.7 mN m(-1). Fourier transform infrared spectroscopy (FT-IR) analysis indicated that the biosurfactant produced by the strain FJAT-14262 was a kind of lipopeptides. Reverse-phase high-performance liquid chromatography (RP-HPLC) and liquid chromatography-mass spectrometry (LC-MS) analysis demonstrated that this lipopeptide contained surfactin with polar amino acids and hydrophobic fatty acid chains. Moreover, bioinformatic analysis revealed that the nonribosomal peptide synthetases genes srfAA, srfAB, and srfAC were structurally conserved in the FJAT-14262 genome. Importantly, the crude surfactant exhibited strong inhibitory activities against Fusarium oxysporum, suggesting that strain FJAT-14262 could be a potential biological control agent against Fusarium wilt.